Detection of Sars-Cov-2 in Wastewater, Using The
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medRxiv preprint doi: https://doi.org/10.1101/2020.06.03.20121426; this version posted June 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 1 Detection of SARs-CoV-2 in wastewater, using the existing 2 environmental surveillance network: An epidemiological 3 gateway to an early warning for COVID-19 in communities 4 5 Salmaan Sharif1 , Aamer Ikram1, Adnan Khurshid1 , Muhammad Salman1, Nayab Mehmood1, 6 Yasir Arshad1, Jamal Ahmad2, Rana Muhammad Safdar3, Mehar Angez1, Muhammad Masroor 7 Alam1 , Lubna Rehman1, Ghulam Mujtaba1, Jaffar Hussain1, Johar Ali1, Ribqa Akthar1, 8 Muhammad Wasif Malik1, Zeeshan Iqbal Baig1, Muhammad Suleman Rana1, Muhammad 9 Usman1, Muhammad Qaisar Ali1, Abdul Ahad1, Nazish Badar1, Massab Umair1, Sana Tamim1, 10 Asiya Ashraf1, Faheem Tahir1, and Nida Ali2 . 11 12 1. National Institute of Health, Islamabad, Pakistan. 13 2. World Health Organization, Islamabad, Pakistan. 14 3. National Emergency Operations Centre for Polio Eradication, Islamabad, Pakistan 15 16 Corresponding Author 17 Salmaan Sharif 18 Department of Virology, 19 Public Health Laboratories Division, 20 National Institute of Health, 21 Islamabad, Pakistan. 22 Email: [email protected] 23 Tel: +92-301-2005000 / +92-51-8442662 (Ext: 110) 1 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. medRxiv preprint doi: https://doi.org/10.1101/2020.06.03.20121426; this version posted June 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 24 Abstract 25 Background: 26 The ongoing COVID-19 pandemic caused by SARs-CoV-2 was transmitted person to person via 27 droplet infections and fecal-oral transmission. This illustrates the probability of environmentally 28 facilitated transmission, mainly the sewage. 29 Method: 30 We used existing Pakistan polio environment surveillance network to investigate presence of 31 SARs-CoV-2 using three commercially available kits and E-Gene detection published assay for 32 surety and confirmatory of positivity. A Two-phase separation method is used for sample 33 clarification and concentration. An additional high-speed centrifugation (14000Xg for 30 min) 34 step was introduced, prior RNA extraction, to increase viral RNA yield resulting a decrease in 35 Cq value. 36 Results: 37 A total of 78 wastewater samples collected from 38 districts across Pakistan, 74 wastewater 38 samples from existing polio environment surveillance sites, 3 from drains of COVID-19 infected 39 areas and 1 from COVID 19 quarantine center drainage, were tested for presence of SARs-CoV- 40 2. 21 wastewater samples (27%) from 13 districts turned to be positive on RT-qPCR. SARs-COV- 41 2 RNA positive samples from areas with COVID patients and COVID 19 patient quarantine 42 center drainage strengthen the findings and use of wastewater surveillance in future. 43 Furthermore, sequence data of partial ORF 1a generated from COVID 19 patient quarantine 44 center drainage sample also reinforce our findings that SARs-CoV-2 can be detected in 45 wastewater. 46 Discussion: 47 This study finding indicates that SARs-CoV-2 detection through wastewater surveillance has an 48 epidemiologic potential that can be used as early warning system to monitor viral tracking and 49 circulation in cities with lower COVID-19 disease burden or heavily populated areas where 50 door-to-door tracing may not be possible. However, attention needed on virus concentration 51 and detection assay to increase the sensitivity. Development of highly sensitive assay will be an 52 indicator for virus monitoring and to provide early warning signs. 2 medRxiv preprint doi: https://doi.org/10.1101/2020.06.03.20121426; this version posted June 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 53 Introduction: 54 Novel coronavirus pneumonia (COVID-19) caused by SARS-Cov-2 infection has become a global 55 emergency through its widespread infection with 6,189,560 confirmed cases resulting 372,469 56 deaths in 213 countries as of 1st June , 2020 (JHU 2020). In December 2019, cluster of 57 pneumonia like disease cases with symptoms including fever, difficulty in breathing, cough and 58 invasive lesion on both lungs were reported from Wuhan, China (WHO 2020a). The causative 59 agent was identified as a Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after 60 ruling out SARS-CoV, MERS-CoV, influenza, avian influenza, adenovirus and other common 61 respiratory pathogens (WHO 2020b). Coronaviruses belonging to family Cornaviridae are 62 enveloped, non-segmented positive sense RNA viruses distributed in human and mammals 63 (Richman DD 2016). However, majority of human coronaviruses have mild infections but two 64 betacornaviruses; severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East 65 respiratory syndrome coronavirus (MERS-CoV) caused outbreaks in last two decades with 10% 66 and 35.6% mortality rate respectively (de Groot et al. 2013; Hui et al. 2004; Ramadan and Shaib 67 2019). 68 . On March 11, 2020 WHO declared it as pandemic, when disease was reported in 114 countries 69 (WHO 2020c). The primary routes of viral transmission (SARS-CoV-2) was considered to be 70 through droplet infections and person to person close contact, but later it is evident from 71 various published studies that there is increasing possibility of fecal-oral transmission (Adhikari 72 et al. 2020; Cheng et al. 2004). This shows the probability of environmentally mediated 73 transmission. Since the early days of pandemic, we got interested in understanding and utilizing 74 the role of environmental sampling mainly the sewage. 75 SARS-CoV-2 resembles 82% with SARS coronavirus (SARS-CoV) which caused an outbreak in 76 2003. Studies have shown the survival of SARS-CoV in stool for up to 4 days (Hui et al. 2004). 77 Another study described the presence of SARS-CoV and its infectious nature in water and 78 sewage for days to weeks (Casanova et al. 2009). It was also described that faulty sewage 79 system contaminated with SARs-CoV in a high-rise housing estate of Hong Kong during 2003 80 was linked to SARS outbreak involving large number of residents of surrounding buildings (Peiris 81 et al. 2003). A recent study highlighted the shedding through stool of SARS-CoV-2 in cluster of 9 3 medRxiv preprint doi: https://doi.org/10.1101/2020.06.03.20121426; this version posted June 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission. 82 nCOVID-19 patients. It was reported that that the RNA concentration decreased from 107 RNA 83 copies/g to 103 RNA copies/g after one week of symptom onset to third week (Roman Woelfel 84 2020). Since the source of transmission of SARS-CoV-2 is still unknown therefore wastewater 85 transmission pathway can become an important mode (UNICEF 2020). Hence, the presence of 86 SARS-CoV-2 in contaminated sewage sample and its role in transmission needs to be 87 investigated. In this study, we used the existing polio environment surveillance network in 88 Pakistan through which sewage samples were collected from designated sites in different 89 districts of the country to investigate presence of poliovirus, its spread and molecular 90 epidemiology. Same samples were processed and tested for detection of SARs-CoV-2 RNA. 91 Methods 92 Untreated wastewater samples (sewage samples) selected for testing in this study were 93 collected using the grab sampling technique. Most of them were those collected for routine 94 polio environment surveillance (ES). Polio ES sites are either open drains or pumping stations 95 and are sampled routinely on monthly basis. Each sampling site represent 100,000 – 300,000 96 population (WHO 2015). Besides, wastewater from drains of some areas with recent history of 97 SARS-CoV2 cases were also collected for detection and re-confirmation of SARs-CoV-2 98 detection. Sampling personnel strictly followed the standard safety guidelines for personnel 99 protective equipment (PPE) required for wastewater sampling. One liter of sewage water was 100 collected from the mid-stream into a sterile, leak proof container at a downstream sampling 101 site during the peak morning flow. These samples were transported in properly sealed 102 container with information form, indicating sampling site, district, sampling date and sampling 103 time, to laboratory within 48 hours of collection maintaining reverse cold chain (WHO 2015). 104 Samples were processed in laboratory for virus concentration using the two-phase separation 105 method (WHO 2003). 106 500 ml of each raw sewage specimens was concentrated. Firstly, clarification of the sample 107 was done by pelleting of larger suspended solids by high speed centrifugation. The clarified 108 sewage sample was mixed with defined amounts of polymers, dextran and polyethylene 109 glycol (PEG). The homogenous mixture obtained by vigorous shaking is left to stand 110 overnight at 4°C in a separation funnel. The polymer helped to form two distinct layers 4 medRxiv preprint doi: https://doi.org/10.1101/2020.06.03.20121426; this version posted June 24, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.